Pharmacokinetics, biodistribution and toxicology of novel cell-penetrating peptides

Cell-penetrating peptides (CPPs) have been used in basic and preclinical research in the past 30 years to facilitate drug delivery into target cells. However, translation toward the clinic has not been successful so far. Here, we studied the pharmacokinetic (PK) and biodistribution profiles of Shuttle cell-penetrating peptides (S-CPP) in rodents, combined or not with an immunoglobulin G (IgG) cargo. We compared two enantiomers of S-CPP that contain both a protein transduction domain and an endosomal escape domain, with previously shown capacity for cytoplasmic delivery. The plasma concentration versus time curve of both radiolabelled S-CPPs required a two-compartment PK analytical model, which showed a fast distribution phase (t1/2α ranging from 1.25 to 3 min) followed by a slower elimination phase (t1/2β ranging from 5 to 15 h) after intravenous injection. Cargo IgG combined to S-CPPs displayed longer elimination half-life, of up to 25 h. The fast decrease in plasma concentration of S-CPPs was associated with an accumulation in target organs assessed at 1 and 5 h post-injection, particularly in the liver. In addition, in situ cerebral perfusion (ISCP) of L-S-CPP yielded a brain uptake coefficient of 7.2 ± 1.1 µl g−1 s−1, consistent with penetration across the blood–brain barrier (BBB), without damaging its integrity in vivo. No sign of peripheral toxicity was detected either by examining hematologic and biochemical blood parameters, or by measuring cytokine levels in plasma. In conclusion, S-CPPs are promising non-toxic transport vectors for improved tissue distribution of drug cargos in vivo.


Results
Pharmacokinetic (PK) studies. Plasma PK profiles of the two S-CPP enantiomers. PK analyses were based on plasma data following a single intravenous (i.v.) injection in the caudal vein of rats (Table 1). Plasma concentrations versus time curves and PK parameters of the S-CPPs L-S-CPP and D-S-CPP are plotted in Fig. 1. In the case of i.v. bolus administration, C max is equal to concentration at time zero (C 0 ), extrapolated from the curve at the y-intercept. As expected, the PK concentration-time pattern of the S-CPPs required a two-compartment model, separating distribution and elimination phases. Plasma counting of 3 H-L-S-CPP or 3 H-D-S-CPP showed a fast distribution phase (T 1/2 α ranging between 1.3 and 3 min) followed by a slower elimination phase (T 1/2 β ranging between 4 and 15 h) after i.v. injection. Central volumes of distribution (V C > 900 mL/kg) were many times more elevated than whole plasma volumes in the rat. These results are consistent with a quick distribution of S-CPPs in organs when injected alone. Finally, although doses, AUC and C 0 were different due to the necessity to inject sufficient amounts of radioactivity to all animals, both S-CPPs had similar T 1/2 , T max , Cl and V C , suggesting no major difference in elimination and distribution.
PK parameters of IgG are not changed by combination with S-CPP.. The PK parameters of 3 H-IgG administered alone or combined with L-S-CPP are shown in Fig. 2. Plasma concentration-time curves were linear and required single-compartment models to estimate the PK parameters. The calculated elimination half-life was over 25 h, consistent with a long blood residence time. The central volume of distribution was closer to plasma volumes in the rat (V C ≈ 65 mL/kg), consistent with retention into the blood compartment. Combination with L-S-CPP did not change the PK parameters measured for each IgG. As expected, the C 0 of L-S-CPP-IgG was higher than IgG due to the difference in dose. Table 1. Experimental design and sample collection. BBB blood brain barrier, D 0 initial dose (in syringe), S-CPP Shuttle-cell penetrating peptides, h hour, IgG immunoglobulin G, IgG antiNUP anti-Nuclear Pore Complex Proteins Antibody, PBS Phosphate-buffered saline. was determined by dividing the radioactivity in each harvested organ, expressed in dpm g −1 , by the radioactivity in the plasma expressed in dpm µl −1 (Fig. 3). Radioactivity from the tritiated S-CPP was detected in all organs 1 h and 5 h after i.v. injection in the tail vein (Fig. 3A,B). The lung, liver, and spleen had the highest levels of S-CPPs for both enantiomers (Fig. 3A,B). This was expected, as CPPs were shown to accumulate in highly vascularized organs 19 . The plasma     Figure 1. Pharmacokinetic profiles of enantiomers L-and D-S-CPP, according to a bicompartment model after a single intravenous injection in rats. Ten-month-old wild-type female Wistar rats were injected in the caudal vein at T 0 and blood was collected at different time points until 24 h. Linear graphical representation of plasma concentrations of 3 H-L-S-CPP (dose = 6.3 × 10 7 dpm kg −1 , or 32.0 µg kg −1 ) (A) and 3 H-D-S-CPP (dose = 16.2 × 10 7 dpm kg −1 , or 3.5 µg kg −1 ) (B). Plasma concentrations are presented as the % of the respective calculated initial concentration (C 0 ). Both sets of curves followed a bicompartment PK model with two phases: a rapid distribution phase (illustrated in green with intercept A as distribution coefficient) and a much longer elimination phase (illustrated in orange with intercept B as elimination coefficient) (C). Data are presented as the mean ± SEM. AUC Area under the curve, C 0 initial estimated concentration, Cl clearance, D 0 initial dose, dpm disintegration per minute, S-CPP Shuttle cell-penetrating peptides, T 1/2α/β half-life of distribution (α) and elimination (β), Vd estimated area of the compound's distribution.  Figure 2. Pharmacokinetic profile of 3 H-IgG combined to L-S-CPP, according to a linear model after a single intravenous injection in rats. Ten-month-old wild-type female Wistar rats were injected in the caudal vein at T 0 and blood was collected at different time points until 48 h. Linear graphical representation of plasma concentrations of 3 H-IgG (at 1.3 × 10 7 dpm kg −1 = 19.9 µg.kg -1 , or with L-S-CPP (1.4 mg kg −1 ). Plasma concentrations are represented as the % of the respective calculated initial concentration (C 0 ). Both sets of curves followed a linear PK model. PK parameters are shown in the inserted Tables. Data are presented as mean ± SEM. Statistical analysis: Student's t-test between 3 H-IgG alone compared to the combination with L-S-CPP at equivalent doses for calculated PK parameters. AUC Area under the curve, C 0 initial estimated concentration, Cl clearance, D i initial theorical dose, dpm disintegration per minute, Dose extrapolated graphically estimated dose, S-CPP Shuttle cell-penetrating peptides, IgG immunoglobulin G, T 1/2 half-life, V D estimated area of the compound's distribution.  www.nature.com/scientificreports/ concentrations of 3 H-L-S-CPP and 3 H-D-S-CPP decreased from 1 to 5 h after i.v. injection. However, the V D was higher at 5 h than at 1 h in several organs such as the brain, heart, muscle, and spleen ( Fig. 3), suggesting a slower clearance in these organs than in the blood. The comparison between L and D enantiomers of S-CPP is shown in Fig. 3C,D. Both enantiomers followed the same pattern at 1 h post i.v. injection, with a predictable accumulation in the liver and spleen. One hour after administration, we observed that the V D of the D form was higher in the heart and liver than the L enantiomer (Fig. 3C). After five hours, higher relative contents of the D form were also found in the plasma and the kidneys. This contrasts with the brain where a higher V D was detected for the L enantiomer. TCA precipitation of tritiated S-CPPs from the liver, the organ that showed the highest accumulation, indicates that 83.1% (± SD 16.4%, n = 3) of the radioactivity measured was still associated with S-CPPs after 5 h.
Combination with S-CPP increases the uptake of 3 H-IgG in the liver. In order to elucidate whether combination with S-CPP alters the biodistribution of a large cargo, we compared the V D in organs 1 h after systemic administration of 3 H-IgG , combined or not to unlabeled L-S-CPP, D-S-CPP, or a scrambled peptide (Fig. 4). Combining  . Apparent volume of distribution (μl g −1 ) of tritiated IgG antiNUP 1 h after co-injection with either forms of S-CPP or a scrambled peptide. Ten-week-old male CD-1 mice were injected in the caudal vein and sacrificed by intracardiac perfusion at 1 h post injection, thereby removing blood from the brain. IgG antiNUP targets nuclear pore proteins. The 3 H-IgG antiNUP dose was 82.5 µg kg −1 ± L/D-S-CPP or combined with a control peptide ("Scramble" or SCR) at 3.6 mg kg −1 . The apparent volume of distribution (μl g −1 ) in each organ was calculated by dividing radioactive counts (dpm g −1 ) of each tissue by plasma counts (dpm µl −1 ). Data are represented on a logarithmic scale with the mean of N = 6-12 ± SEM. Statistical analyses were performed on values after logarithmic transformation by a One-Way ANOVA parametric test followed by a Tukey post-hoc test (**p < 0.01; ***p < 0.001; ****p < 0.0001), a Welch ANOVA parametric test followed by a Dunnett's post-hoc test (¤ p < 0.05; ¤¤¤ p < 0.001). ns not significant, dpm disintegration per minute, S-CPP Shuttle cell-penetrating peptides, IgG immunoglobulin G, V D estimated area of the compound's distribution. www.nature.com/scientificreports/ the 3 H-IgG with D-S-CPP led to significantly higher plasma concentrations at 1 h post injection, when compared to 3 H-IgG combined with the scrambled control peptide. This effect was not significant for L-S-CPP. The combination of 3 H-IgG antiNUP to S-CPPs increased its V D in the liver but decreased its V D in the muscle (Fig. 4). A higher relative distribution was also seen in the spleen but only for D-S-CPP (Fig. 4). No other significant difference was measured in other organs (Fig. 4). These data suggest that S-CPPs induced a preferential distribution of IgG in the liver and possibly spleen, but had the opposite effect in the muscle.

Transport of L-S-CPP across the blood-brain barrier (BBB).
The biodistribution studies revealed a V brain of 45.6 ± 13.1 µl.g −1 and 715.1 ± 335.0 µl g −1 , at 1 h and 5 h respectively, for 3 H-L-S-CPP (Fig. 3), consistent with a relative cerebral accumulation of 3 H-L-S-CPP. To better characterize the passage of this CPP through the BBB, we performed ISCP to infuse directly 3 H-L-S-CPP into the carotid at a dose of 0.2 μg corresponding to a concentration of 0.08 μg/ml. The observed brain coefficient uptake (Clup) of 3 H-L-S-CPP (7.2 ± 1.8 µl g −1 s −1 ) indicates a relatively high capacity of S-CPPs to cross the BBB (Fig. 5A). To gain further insights on the mechanism of transport, we used increasing concentrations of 3 H-L-S-CPP (0.08, 0.16 and 0.32 μg/ml). The absence of saturation in the rate of transport across the BBB (Fig. 5A) suggests that L-S-CPP transport across the BBB probably does not involve receptor-mediated endocytosis but mostly simple diffusion. The total pmol/g of 3 H-L-S-CPP collected in the brain increased linearly with rising concentrations of 3 H-L-S-CPP perfused (r 2 = 0.88, p < 0.0001; Fig. 5B), which also indicates that L-S-CPPs do not use a saturable mechanism. Moreover, the extravascular % of 3 H-L-S-CPP was 97.5 ± 0.3%, which is consistent with a fast distribution outside of the vascular compartment (Fig. 5C). Finally, the Vvasc measured in each mouse using 14 C-sucrose remained in the normal range (lower than 20 µl g −1 ), confirming that the CPPs did not alter the integrity of the BBB.
Toxicology studies. To assess their toxicity, we have injected high doses of L-S-CPP and D-S-CPP (each at 3.6 mg kg −1 ) in the tail vein of mice, BID for 5 days. Mice did not lose weight during this time period. A blood sample was collected at the end of the experiment for each animal and hematological and biochemical analyses were performed to evaluate toxicity. The data revealed no abnormalities, as observable differences between mice remained within normal ranges (Tables 2 and 3). Serum cytokine quantification using multiplex ELISA was also evaluated. A large panel of cytokines were measured in triplicates in plasma samples: TNF-α, IFN-ɣ, GM-CSF, IL-1ß, IL-2, IL-5, IL-4 and IL-10. Most pro-inflammatory cytokines or anti-inflammatory cytokines were below detection threshold. Therefore, despite the high doses used (in the mg/kg range), S-CPPs did not induce systemic inflammation or immunogenicity (Table 3).

Discussion
Many potential therapeutic targets are located inside of cells; however, these targets often remain out of reach because therapeutic molecules must cross several physiological barriers, including ultimately the cytoplasmic membrane. The central nervous system (CNS) is particularly well protected by the blood-brain barrier (BBB),

A B
Extravascular (%) = 97.5 ± 0.20  www.nature.com/scientificreports/ which blocks most biopharmaceuticals. For some decades, CPPs have been proposed as vehicles for intracellular delivery and solutions for this pharmaceutical challenge. The emergence of CPPs in the clinic has been impeded by the lack of preclinical PK-BD data. The aim of the present study was to investigate PK and BD parameters of CPPs alone or combined in vivo as well as their toxicity and brain uptake. After intravenous administration, we observed that both S-CPPs displayed a fast distribution phase of less than 3 min. This is consistent with a quick distribution in organs due to the small size and relative lipophilicity of the CPPs 19 . Although a significant portion was rapidly removed from the blood, S-CPPs then displayed a longer and slower elimination phase (T 1/2 over 450 min), whichwas uncovered because of the use of a two-compartment PK model. True in vivo PK studies of CPPs in the literature are scarce. For example, one study using a panel of ten CPPs report half-lives ranging from 72 (penetratin) to 528 (TAT) min to over 72 h by measuring their stability in vitro at 37 °C in human serum 19 , which does not take into account tissue distribution and metabolism, as well as excretion. On the other hand, Lee et al. reported T 1/2 values of 0.87 and 107 min for the distribution and elimination phases, respectively, after i.v. injection of TAT-biotin in rats 19,40,41 . The present results are in line with these studies and suggest that S-CPPs alone may display a sufficient residence time to exert a pharmacological effect in target tissue.

Extravascular % of 3 H-L-S-CPP
Very few studies have compared CPP enantiomers in vivo 19,22,42,43 . The rationale for their comparison here was that stereoselective interactions with liver enzymes or the cell membrane might differ between L-S-CPP and D-S-CPP 44,45 . For example, it has been postulated that D-form CPPs may be more resistant to degradation by enzymes compared to L-forms, thereby increasing their stability in vivo 38,39 . Here, the D-form exhibited slightly lower T 1/2 and AUC, as well as higher clearance and V c , compared to the L-form, although these differences did not reach statistical significance. Thus, our results do not suggest obvious advantages of using either enantiomer to improve PK parameters, although biodistribution was affected (see below).
As S-CPPs are designed to deliver protein-based cargo, PK parameters of intravenously injected immunoglobulin G (IgG), in the presence or not of S-CPPs, were investigated. As expected, the calculation of PK parameters of IgG required a single-compartment model because of the slow distribution and stability of IgG in the blood in vivo. Comparison of IgG alone or combined to L-S-CPP did not reveal significant differences in PK parameters such as AUC, clearance or V D . This suggests that the combination to CPPs did not modify cargo functionality. Thus, the PK data obtained in this study is consistent with what is expected for IgG, with a half-life of days to weeks in the mouse 46,47 . Interestingly, with regards to biodistribution, D-S-CPP increased the plasma concentration of IgG 1 h post-injection in mouse when compared to the scramble peptide. This indicates that IgG/S-CPP formulations may present an advantageous circulating time, increasing the propensity to reach their intracellular target after systemic administration.
The biodistribution studies provided a comparative view of the concentration of S-CPP reaching different organs at two different time points selected during the elimination phase. The apparent volume of distribution increased between 1 and 5 h post-injection in many organs, suggesting a relative accumulation over time. While this was true for both enantiomers in the heart and muscle, L-S-CPP displayed a preferential relative distribution in the brain and spleen, whereas for D-S-CPP it was the liver and pancreas. The comparison of the two Table 2. Blood hematology parameters after chronic high dose of S-CPP. Mice were injected i.v. twice a day for 5 days (total of 10 injections) and sacrificed by intracardiac perfusion. Injected dose was 3.6 mg kg −1 . Data are presented as the mean ± SEM. The statistical analysis did not reveal any significant difference.

PBS (n = 5)
L-S-CPP (n = 7) D-S-CPP (n = 8) www.nature.com/scientificreports/ enantiomers at 5 h shows that the D form was relatively more concentrated in the plasma, heart, liver and kidneys than the L form. This is consistent with previous work suggesting a greater stability of D-CPPs in vivo 39 and suggests that the enzymatic resistance of D amino acids is noticeable only after several hours. A notable exception was the brain, in which the L-S-CPP exhibited a higher apparent volume of distribution than the D-form at 5 h post-injection. This tells us that, despite a reduction in plasma levels of both L-S-CPP and D-S-CPP between 1 and 5 h, concentrations in key organs remained at appreciable levels at both time points. For example, based on values in dpm/g and specific activity in dpm/µg, concentrations of S-CPP in the liver ranged between 2 and 10 nM up to 5 h post injection. The integrity of the S-CPPs (> 80%) in the liver was confirmed using TCA precipitation. Even in the brain, the levels of L-and D-S-CPP reached the low nanomolar zone (0.17 and 0.02 nM, respectively). Overall, the data presented indicates that S-CPPs can reach therapeutically relevant concentrations in target organs after systemic administration. In future clinical applications, S-CPPs are unlikely to be utilized alone and their efficacy will ultimately be determined by the ability of their cargo to reach target organs 22,32 . The biodistribution of IgG antiNUP was investigated with or without combined S-CPPs or a scrambled peptide. The combination with D-S-CPP increased plasma concentrations of the IgG at 1 h post-injection, suggesting a slower elimination not apparent in the previous PK experiment. The most striking observation was that both S-CPPs induced a preferential distribution of IgG in the liver but had the opposite effect in the muscle. A higher accumulation of IgG combined with D-S-CPP was also observed in the spleen compared to the scramble peptide. There is published evidence that D-penetratin improved nasal absorption of interferon beta (IFN-β) better than the L form after intranasal administration 48 . Here, concentrations of cargo IgG were estimated to be between 0.3 and 0.5 nM in the liver and spleen 1 h after administration. It should be reminded, however, that combining a CPP to a cargo smaller than an IgG may have had more impact on its distribution. Nevertheless, such an increase in accumulation of S-CPP/IgG complexes in the liver and spleen, while avoiding the muscle, may be therapeutically valuable for certain indications.
Considering their smaller size (up to 30 amino acids in length), cationic and/or amphipathic CPPs have a greater potential to penetrate the BBB than other transport systems 49 . Previous studies have suggested that most CPPs do not have access to the CNS 14 , in part because of their low AUC 41 . Here, we observed that L-S-CPP Table 3. Blood biochemical parameters and concentrations of pro-and anti-inflammatory cytokines after chronic high dose of S-CPP. Mice were injected i.v. twice a day for 5 days (total of 10 injections) and sacrificed by an intracardiac perfusion. Normal levels of γGT (γ Glutamyl Transferase, U/l) and TB (total bilirubin, µmol/l) do not exceed 10, whereas creatinine should not go over 18 µmol/l. Injected dose was 3.6 mg kg −1 . Biochemistry data are presented as the mean ± SEM, and cytokines as the mean of duplicates (PBS) or triplicate (L/D-S-CPP) in pg/ml when it was possible because most data were out of range (OOR) and extrapolated by the software. Statistical analysis: Unpaired t-test ( £ p < 0.05, ££ p < 0.01; ££££ p < 0.0001) or Mann-Whitney ( $$ p < 0.01; $$$ p < 0.001), PBS versus S-CPP injected. The differences measured remained within normal ranges for a mouse.  www.nature.com/scientificreports/ accumulated preferentially in the brain at 1 and 5 h post-injection. Using in situ cerebral perfusion (ISCP) to directly assess its capacity to cross the BBB, we calculated a Clup of approximately 7 µl g −1 s −1 . For the sake of comparison, a control IgG, an IgG binding the transferrin receptor (receptor-mediated endocytosis), glucose (facilitated transport) and diazepam (passive diffusion) display Clup of ≈0.005, ≈0.03, ≈1 and ≈40 µl g −1 s −1 , respectively 47,[50][51][52] . Importantly, the infusion of 0.8 μg/mouse (80 nM) of CPP into the carotid did not impair the integrity of the BBB. The results from ISCP experiments showed that the rate of transport of L-S-CPP is relatively high and maintained or even increased with escalating concentrations. This indicates that the transport of CPPs across the BBB is not saturable and could be explained at least in part by free diffusion. The data is also consistent with a saturable efflux of L-S-CPP back to the blood. However, as most CPPs penetrate cells through more than one mechanism 53,54 we cannot exclude that S-CPP use specific transport mechanisms in the presence of a cargo or under other experimental conditions. This is consistent with the hypothesis that S-CPPs activate translocation and endocytosis in a dose-dependent manner 32 . In addition, S-CPPs show affinity for heparan sulfate proteoglycans 32 , a type of membrane-bound entity present in endothelial cells of the BBB 55 . Further studies are needed to determine the exact mechanism of transport into the brain.
Owing to their physicochemical properties, CPPs can be internalized by almost any type of cell. However, few studies have addressed the toxic and immunological responses to CPPs in vivo. Here, hematologic and cytokine endpoints did not reveal any difference between treatment and control groups. Regarding biochemistry parameters, levels of alanine transaminase for both treated groups were higher than controls. L-S-CPP-injected mice also exhibited higher levels of albumin compared to phosphate-buffered saline (PBS)-injected mice. Yet, all of these levels remained in the normal range for a mouse 56 . In addition, no animal exhibited obvious changes in physical appearance, activity level, or body weight. Although no signs of toxicity were observed in the present study, previous studies have suggested that CPPs might act as double edge swords mediating a wide variety of unpredictable biological effects 38,[57][58][59] . For this reason, we cannot fully rule out potential toxicity despite the high doses used in this study.
In summary, S-CPPs PK, biodistribution and toxicity data gathered here argue in favor of their potential use in vivo, particularly when combined to cargos with long residence time in circulation. Therapeutically relevant distributions of S-CPPs were reached in multiple organs, such as the liver, the spleen and the kidney, but also the CNS for uncombined L-S-CPP. The present results therefore suggest that the capacity of S-CPPs to improve target engagement of biopharmaceuticals after systemic administration should be further investigated.

Methods
Materials and radioactive labelling. Peptides used in this study were synthesized by GL Biochem (Shanghai, China), as described 32,33,60,61 . They performed the purification by reversed-phase high performance liquid chromatography and confirmed peptide identity by mass spectrometry (Agilent-6125B). Purity reached 95%. L-S-CPP and D-S-CPP are the two enantiomers of S-CPP with the following properties: Peptide sequences are proprietary to Feldan Therapeutics 60 and include a protein transduction domain (PTD) and an endosomal escape domain (EED) (Patent No.; International Publication Number, WO2022204806A1, WO2022082315A8, WO2017175072A1, WO2022077121A1). Tested cargos were immunoglobulins G (IgG, molecular weight ≈ 150 000 g mol −1 ). We used a mouse monoclonal IgG recognizing intracellular anti-nuclear pore complex proteins (IgG antiNUP ) purchased from BioLegend, as well as a rat IgG possessing no mammalian reactivity (IgG control ) purchased from Bio-X-Cell. CPPs and cargoes were co-incubated for at least 30 min in PBS to achieve non-covalent combination.
L-S-CPP and IgGs were radiolabeled with N-Succinimidyl propionate-2,3- www.nature.com/scientificreports/ agreement with suggestions from our Animal Ethics Committee. Rats (10-month-old females) were anesthetized with isoflurane and injected in the caudal vein at T 0 . Blood samples were collected at the jugular vein at different time points until 24 or 48 h (Table 1). Blood samples were centrifuged, and plasma was counted for both total and TCA-precipitable radioactivity per volume. Rats were sacrificed using a carbon dioxide (CO 2 ) chamber. Injected doses were: 3 H-L-S-CPP = 6.3 × 10 7 dpm kg −1 (32.0 µg kg −1 ) and 3 H-D-S-CPP = 16.2 × 10 7 dpm kg −1 (3.5 µg kg −1 ) and 3 H-IgGs = 1.3 × 10 7 dpm kg −1 (18.5-19.9 µg kg −1 ) ± non-radiolabeled L-S-CPP (1.4 mg kg −1 ). Injected doses were selected to provide significant detection in counting disintegrations per minute (dpm) well above background measures. Observation of the plasma-level time curve indicated that CPPs declined biexponentially following a twocompartment model kinetic with 2 phases: distribution (fast initial decline in blood concentrations) and elimination (slower subsequent decline in blood concentrations) (Fig. 1). By contrast, IgGs plasma-level time curve was linear following an i.v. injection, where the i.v. bolus entered the bloodstream directly and declined linearly as a single first-rate process (Fig. 2).
In the biexponential model used for S-CPPs, C 0 was extrapolated after a logarithm transformation of the distribution data while, for IgG, C 0 was extrapolated from the linear curve at the y-axis intercept. Of note, after an i.v. injection, C 0 is equivalent to C max . The other PK parameters such as the apparent distribution and elimination half-life (T 1/2 α,T 1/2 β), plasma clearance (Cl), central volume of distribution (V C = D 0 /C 0 ), and the area under the concentration versus time curve (AUC) 62,63 were generated using Excel add-in PK Solver 64 .
Linear kinetic of IgG obtained from the plasma-level time curve after i.v. bolus, is best described by Eq. (1) mathematical expression, where ke represents the distribution rate constant dependent on the amount or concentration of IgG present, and C 0 the initial IgG concentration.
Biexponential kinetic of S-CPPs followed a two-compartment model corresponding to Eq. 2, where C(t) is the plasma concentration at time t; A and B are intercept terms (distribution and elimination coefficients which exhibit two compartments); α is the distribution rate constant; and β is the elimination rate constant 65,66 . Biodistribution. Mice (10-week-old males weighing ≈40 g) were injected in the caudal vein at T 0 and sacrificed by cold-PBS intracardiac perfusion 1 or 5 h post injection under deep anesthesia with ketamine/xylazine intraperitoneal (i.p.) injection (300 mg kg −1 ketamine, 30 mg kg −1 xylazine). Mice were used because our Animal Ethics Committee encourages the use of the smallest animal species possible and there was no methodological advantage in using rats. The following preparations were compared: tritiated S-CPP in its 2 enantiomeric forms, 3 Table 2. The scrambled peptide had 30 amino acids, like S-CPPs, but without any CPP property.
Samples from organs (brain, lung, heart, liver, spleen, pancreas, gastrocnemius muscle and kidney) with an approximate weight of 150 mg were collected, weighed and solubilized with SOLVABLE at 50 °C overnight for quantification of tritiated molecules. To prevent quenching, most organs (lung, heart, kidney, spleen and liver) were incubated with 30% hydrogen peroxide (H 2 O 2 ) (200 µL) after solubilization. Blood samples were centrifuged, and both plasma and organs were counted after addition of 9 ml of Ultima Gold scintillation cocktail. Apparent distribution volumes (V D ) in µl g −1 were computed by dividing the concentration in organs relative to weight (dpm g −1 ) by the concentration in the blood relative to volume (dpm µl −1 ). The use of V D allowed data normalization with plasma concentration for each animal. TCA-precipitated radioactivity ranged between 90 and 100% in blood samples after 1 h.
Toxicology. Mice (10-week-old male CD-1) were injected bis in die (BID) during 5 days, in the morning and the afternoon with a gap of 6 h between the two injections, alternately into the tail vein and the retro-orbital region ( Table 1). The injected dose was 3.6 mg kg −1 for each form of S-CPP. Animals were sacrificed by cold-PBS intracardiac perfusion. Blood hematology and biochemical parameters were determined at our hematology facilities (Heska Element/Dri-Chem). To assess pro-and anti-inflammatory cytokines, Bioplex kits for 8 cytokines were used (Biorad Mouse Cytokine 8-plex Assay #M60000007A).
In situ cerebral perfusion to assess the passage through the blood-brain barrier (BBB).. The in situ cerebral perfusion (ISCP) technique allows to measure the volume of distribution in the brain and transport coefficient (Clup) across the BBB of compounds after an intracarotid perfusion. Since 100% of the perfusate reaches the BBB, distribution and transport parameters can be readily determined. ISCP was conducted as previously described [67][68][69][70] . Briefly, 4-month-old male balb/c mice were anesthetized by intraperitoneal injection of xylazine/ketamine (8/140 mg kg −1 ). The right common carotid artery was catheterized to perfuse 0.2-0.8 µCi ml −1 of 3 H-L-S-CPP (corresponding to doses of 0.2, 0.4 and 0.8 µg/mouse and concentrations of 0.08, 0.16 and 0.32 µg/ml) at 2.5 ml min −1 in a bicarbonate buffered physiological saline, co-perfused with 14 C-sucrose (0.12 µCi ml −1 ) to quantify the vascular space and to assess the physical integrity of the BBB. The measured vascular space remained under 20 µl g −1 in the present work confirming physical integrity of the BBB. The perfusion was terminated by decapitating the mouse at selected time (60 s). The right cerebral hemispheres and  www.nature.com/scientificreports/ aliquots of the perfusion fluid were collected and weighed. Tissue samples were digested in 1 mL of Solvable at 50 °C overnight, and then cooled to room temperature and mixed with 9 mL of Ultima Gold scintillation cocktail. Both isotopes were counted in a Hidex 300 SL Liquid Scintillation Counter. The brain transport coefficient (Clup, μL g −1 s −1 ) of 3 H-L-S-CPP was calculated from the measured volume of distribution (V brain , µl g −1 ) of 3 H-L-S-CPP, corrected with the vascular space (V vasc , μl g −1 ) determined with 14 C-sucrose. The following equation was used: V brain (µl g −1 ) represents the apparent volume of distribution of study compound, T (s) is the time, X (dpm g −1 ) is the quantity of radioactivity found in the brain corresponding to the injected molecule and C is the concentration (dpm µl −1 ) in the perfusion fluid.
In addition, extravascular % of 3 H-L-S-CPP corresponding to the fraction not remaining in the vascular compartment was estimated following this equation: Statistical analysis. Data are shown as mean ± standard error of the mean (SEM). When normality was verified, unpaired Student's t-tests were used to identify significant differences between two groups. Otherwise, non-parametric Mann-Whitney tests were performed. Statistical differences between three groups or more were determined using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons tests or, when variances were not equivalent according to a Bartlett's test, Welch-ANOVA followed by Dunnett's post-hoc tests. Statistical analysis of biodistribution data were performed after logarithmic transformation. All of the tests were two-tailed, and statistical significance was set as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Data availability
The datasets generated during this study are available from the corresponding author on reasonable request.